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American Journal of Clinical Nutrition, Vol. 88, No. 4, 986-996, October 2008
© 2008 American Society for Nutrition

ORIGINAL RESEARCH COMMUNICATION

Portal infusion of amino acids is more efficient than peripheral infusion in stimulating liver protein synthesis at the same hepatic amino acid load in dogs1,2,3

Dominique Dardevet, Scot R Kimball, Leonard S Jefferson, Alan D Cherrington, Didier Rémond, Catherine A DiCostanzo and Mary Courtney Moore

1 From the Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN (DD, ADC, CAD, and MCM); INRA, Unité de Nutrition Humaine, UMR1019, Saint Genès Champanelle, France (DD and DR); and the Department of Cellular and Molecular Physiology, Penn State University College of Medicine, Hershey, PA (SRK and LSJ)

2 Supported by NIH grant RO1 DK 43706 (to ADC) and Vanderbilt Diabetes Research and Training Center grant SP-60-AM20593 and NIH grant DK-13499 (to LSJ).

3 Address reprint requests to D Dardevet, INRA UMR 1019 Unité de Nutrition Humaine, Saint Genes Champanelle, 63122 Ceyrat, France. E-mail: dardevdp{at}clermont.inra.fr.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: Hepatic glucose uptake is enhanced by the portal delivery of glucose, which creates a negative arterioportal substrate gradient. Hepatic amino acid (AA) utilization may be regulated by the same phenomenon, but this has not been proven.

Objective: We aimed to assess hepatic AA balance and protein synthesis with or without a negative arterioportal AA gradient.

Design: Somatostatin was infused intravenously, and insulin and glucagon were replaced intraportally at 4- and 3-fold basal rates, respectively, in 3 groups (n = 9 each) of conscious dogs with catheters for hepatic balance measurement. Arterial glucose concentrations were clamped at 9 mmol/L. An AA mixture was infused intravenously to maintain basal concentrations (EuAA), intraportally to mimic the postmeal AA increase (PoAA), or intravenously (PeAA) to match the hepatic AA load in PoAA. Protein synthesis was assessed with a primed, continuous [14C]leucine infusion.

Results: Net hepatic glucose uptake in the PoAA condition was ≤50% of that in the EuAA and PeAA conditions (P < 0.05). In the PoAA and PeAA conditions, hepatic intracellular leucine concentrations were 2- to 2.5-fold those in the EuAA condition (P < 0.05); net hepatic leucine uptake and [14C]leucine utilization were {approx}2-fold greater (P < 0.05) and albumin synthesis was 30% greater (P < 0.05) in the PoAA condition than in the EuAA and PeAA conditions. Phosphorylation of ribosomal protein S6 [downstream of the mammalian target of Rapamycin complex 1 (mTORC1)] was significantly higher in the PoAA, but not PeAA, condition than in the EuAA condition.

Conclusions: Portal, but not peripheral, AA delivery significantly enhanced hepatic protein synthesis under conditions in which AAs, glucose, insulin, and glucagon did not differ at the liver, an effect apparently mediated by mTORC1 signaling.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Protein synthesis in the liver and intestines is considerably higher per gram of tissue than in most other tissues and accounts for 25% of whole-body protein synthesis (1). Forty percent of the protein synthesized in the liver is secreted, with albumin alone accounting for almost 40% of secreted proteins. After a mixed meal, the liver is exposed to high levels of amino acids (AAs) and insulin. In adult humans, increasing AA concentrations by intravenous infusion results in a stimulation of protein synthesis across the splanchnic bed (2).

Splanchnic metabolism includes both gut and hepatic metabolism, for which regulatory pathways could differ. Studies conducted in mature animals with intravenous infusion of AAs have shown that AAs and insulin have no stimulatory effect on liver protein synthesis (3-5). Conversely, in neonates, hepatic protein synthesis can be stimulated by the rise in AAs (6, 7). Davis et al (7) observed a developmental decline in AA-induced stimulation of hepatic protein synthesis in growing animals. Similarly, aging is associated with a decline in the anabolic response of skeletal muscle to food intake and AAs (8-10). From these studies, one might infer that the sensitivity of the liver to AAs decreases during aging. However, this inference is inconsistent with findings in adult human volunteers, in whom dietary AAs were shown to increase hepatic secretory protein synthesis (11-14).

One difference between the animal and human studies cited above is the route of administration of AAs: peripherally in most of the animal studies versus orally in most of the human investigations. We hypothesized that, in the mature organism, the route of delivery of AAs is critical in the stimulation of hepatic protein synthesis. In support of this hypothesis, human studies have shown that albumin synthesis is stimulated with oral meal feeding, whereas it is not responsive to intravenous nutrients (4, 12). Furthermore, we have shown that the net hepatic uptake of glutamine and the net fractional extractions of glutamine and serine are significantly increased during the portal vein administration of AAs compared with a peripheral infusion matching hepatic AA loads (15). From the latter study, it is also tempting to hypothesize that the portal delivery of the AAs directed the AAs to hepatic protein synthesis. We therefore designed the present study to compare, in conscious adult dogs, the rate of hepatic protein synthesis observed during portal or peripheral delivery of an AA mixture that reproduces the normal elevation of AAs seen in the portal vein postprandially. Because protein metabolism is sensitive to AA availability, the concentration and the infusion rate of the AA mixtures were adjusted to maintain the same hepatic AA load between groups under pancreatic clamp conditions. The synthesis rate of resident or exported proteins was then assessed, as was the intracellular signaling pathway leading to protein synthesis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and surgical procedures
Experiments were performed on 42-h fasted, conscious, adult male mongrel dogs (20–25 kg; n = 27) that had been fed once daily a standard meat and kibble diet (31% protein, 52% carbohydrate, 11% fat, and 6% fiber based on dry weight; Kal Kan, Vernon, CA, and Purina Lab Canine Diet no. 5006, Purina Mills, St Louis, MO). The dogs were housed in a facility that met the animal care guidelines of the American Association for the Accreditation of Laboratory Animals, and all protocols were approved by the Vanderbilt University Medical Center Animal Care Committee. At least 16 d before experimentation, a laparotomy was performed with the animals under general anesthesia. Silastic catheters (Dow Corning, Midland, MI) for blood sampling were placed into the portal vein, a hepatic vein, and a femoral artery, and infusion catheters were inserted into a jejunal vein and a splenic vein as previously described (16). Ultrasonic flow probes (Transonic Systems, Ithaca, NY) were placed around the portal vein and hepatic artery. On the day of the experiment, the catheters were exteriorized under local anesthesia, and intravenous access was established in 3 peripheral veins. Dogs were used for an experiment only if they met established criteria for good health (17).

Experimental design
As shown in Figure 1Go, in each of the 3 groups (n = 9 dogs each), the protocol consisted of an equilibration period (0–90 min) and a basal period (90–120 min), followed by an experimental period (120–300 min). At time (t) = 0 min, a continuous infusion of indocyanine green dye (0.08 mg/min; Sigma Chemical, St Louis, MO) was started. At t = 120 min, the timer was stopped briefly. A peripheral venous infusion of somatostatin (0.8 µgkg–1min–1) was begun to inhibit endogenous pancreatic hormone secretion. Two minutes later, an intraportal insulin infusion (1.2 mUkg–1min–1; to produce 3- to 4-fold basal circulating concentrations) was initiated. Two minutes after the insulin infusion started, an intraportal glucagon infusion (1.65 ngkg–1min–1; to produce 3-fold basal concentrations) was started. Two minutes later, dextrose and AA infusions were initiated, and the timer was restarted. Over many years, we have shown that this staggered approach to initiating infusions is effective in controlling insulin and glucagon concentrations.


Figure 1
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  		FIGURE 1.. Experimental design. AA, amino acid; ICG, indocyanine green dye.*HGL, hepatic glucose load; **same amino acid hepatic load.

 
A dextrose solution was infused peripherally at variable rates starting at t = 120 min to clamp the arterial plasma glucose concentration at 9.0 mmol/L. The infusion rate of glucose was adjusted in response to the plasma glucose concentration, which was measured every 5 min. The euaminoacidemia group (EuAA) received a mixture of AAs peripherally to maintain the plasma AAs at their postabsorptive concentrations, because insulin is known to decrease plasma AA concentrations; the portal group (PoAA) received AAs intraportally to mimic a meal; and the peripheral group (PeAA) received AAs peripherally. Because protein metabolism is sensitive to AA availability, concentrations of the AA mixtures were adjusted to maintain hepatic AA load equivalence for each amino acid in the PoAA and PeAA groups. AA concentrations of the infusates (Table 1Go) were determined in pilot experiments, and the mixture was infused at the rate of 0.034 mLkg–1min–1. Protein synthesis was assessed in all groups by a primed (7.6 µCi/kg), continuous infusion (0.76 µCikg–1min–1) of [U-14C]leucine (Moravek Biomedicals, Brea, CA) beginning at 120 min. Blood was collected in tubes containing EDTA. At the end of the 300-min experimental period, animals were killed, and samples of each liver lobe were rapidly freeze clamped and stored at –80 °C.


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  		TABLE 1. Concentration of the amino acid mixture infused in the 3 experimental groups1

 
Analytic procedures
Plasma glucose, insulin, and glucagon concentrations were measured as previously described (18). Plasma amino acids were analyzed by reversed-phase HPLC of their phenyl isothiocyanate derivatives (PicoTag; Waters, Woburn, MA). Free and bound leucine-specific radioactivities were determined as described by Donahue et al (19). Plasma albumin was isolated by ethanol extraction from trichloroacetic acid–precipitated plasma proteins as described previously (4).

Methods of calculation
Hepatic load (µmolkg–1min–1)
The load of a substrate or hormone reaching the liver (HL) was calculated as

Formula 1(1)
where Fa and Fp are the arterial and portal flows (mLkg–1min–1), and Ca and Cp are the concentrations of the variable in question in arterial and portal blood or plasma, as appropriate.

Estimated hepatic plasma sinusoidal hormone concentrations were calculated as HL/total hepatic plasma flow.

Net hepatic substrate balance (nmolkg–1min–1)
Whole-blood glucose concentrations and plasma AA concentrations were used, along with blood or plasma flow, as appropriate, to assess net hepatic substrate balance (NHSB). NHSB was determined as

Formula 2(2)
where Fh represents blood or plasma flow in the hepatic vein (the sum of Fa and Fp) and Ch represents the substrate concentrations in the hepatic vein. A positive value represents net production, and a negative value represents net uptake of the substrate by the liver.

Hepatic leucine uptake (nmolkg–1min–1)
Calculations were based on the differences between the specific radioactivity (Sa) of [14C]leucine entering and exiting the liver (Sain and Sah) and the tracee concentration across the liver (Cin and Ch).

Formula 3(3)
where Saart and Saport are the hepatic artery and portal vein leucine specific activities, and Fart and Fport are the hepatic and portal vein blood flows, respectively.

Formula 4(4)
where Cart and Cport are the hepatic artery and portal vein tracee concentrations.

Formula 5(5)

Formula 6(6)
where Sa liver is the liver intracellular specific radioactivity of the tracer.

Protein synthesis rate
Protein synthesis was calculated by using the continuous infusion of [14C]leucine as a tracer. Briefly, a primed, continuous infusion of [14C]leucine was used to rapidly reach a [14C]leucine Sa plateau in plasma. A single sample of the product (liver proteins or albumin) was then used and not the incremental increase in specific activity of protein-bound leucine across multiple time points. Liver protein and albumin synthesis rates (Ks in %/d) were calculated by using the following equation:

Formula 7(7)
where Sab and Satissue are the specific radioactivities at time t of the protein-bound leucine and the specific radioactivity of the leucine precursor pool between time 0 and time t. The Sa of the tissue fluid was used as the Sa of the precursor pool, because Ahlman et al (5) showed it to be a reliable surrogate measure of leucyl-tRNA specific radioactivity (the real precursor pool for protein synthesis) in a swine model. Protein synthesis was measured in the 7 lobes of the liver, because it appeared from our preliminary studies that slight differences in the rate of protein synthesis might occur among lobes. Because labeled albumin appears in the bloodstream with a lag time of 20 min after the beginning of the tracer infusion (secretion time determined in a pilot experiment), the calculation of albumin synthesis was identical to that of protein synthesis except that it was calculated with a t = t – 20, 20 representing the average excretion time in minutes of newly synthesized plasma proteins by the liver.

Measurement of protein phosphorylation
Phosphorylation of ribosomal protein (rp) S6 on Ser235/236 and Ser240/244 and of eukaryotic initiation factor 4G (eIF4G) on Ser1108 was measured by Western blot analysis as described previously (20) with anti-phospho-rpS6(Ser235/236) and anti-phospho-rpS6(Ser240/244) antibodies or anti-phospho-eIF4G(Ser1108) antibodies, respectively (Cell Signaling Technology, Beverly, MA). Phosphorylation of ribosomal protein S6 kinase 1 (S6K1) and eIF4E binding protein 1 (4E-BP1) was measured as changes in migration during gel electrophoresis as described previously (20). Briefly, frozen liver samples were homogenized in 7 volumes of buffer consisting of 20 mmol HEPES/L (pH 7.4), 100 mmol KCl/L, 0.2 mmol EDTA/L, 2 mmol EGTA/L, 50 mmol NaF/L, 50 mmol β-glycerophosphate/L, 0.1 mmol phenylmethylsulfonyl fluoride/L, 1 mmol benzamidine/L, and 0.5 mmol sodium vanadate/L with a Polytron homogenizer. The homogenate was centrifuged at 1000 x g for 3 min, and the supernatant was subjected to Western blot analysis by using antibodies that recognize the protein only when it is phosphorylated on specific amino acid residues (rpS6 and eIF4G) or antibodies that recognize the protein regardless of phosphorylation state.

Statistical analysis
Data are expressed as means ± SEs and were analyzed by use of XLSTAT (Addinsoft, New York, NY; version 7.5.2). For net hepatic glucose balance, albumin synthesis, blood flows, and sinusoidal hormone concentrations, the statistical evaluation of the data was performed by a 2-way repeated-measures analysis of variance to test the group and time effects and time x group interaction. When significant, the Bonferroni test was used for post hoc analysis. Differences were considered significant when P < 0.05.

For protein metabolism (hepatic leucine balance, hepatic leucine utilization, and hepatic protein synthesis rates), statistical evaluations of the data were performed by one-way ANOVA to analyze the effect of the route of delivery of AAs. Within the same animal of each group, values were calculated as the mean of individual values recorded between 210 and 300 min. When a significant overall effect was detected, differences among individual means were assessed with the Bonferroni test to determine significant differences. Differences were considered significant when P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Hormone concentrations, blood flows, and glucose balance data
Arterial blood flows were not significantly different between groups but increased slightly during the experimental period. Portal blood flows were also not significantly different between groups but significantly decreased during the experimental period (Table 2Go). As a result, total hepatic blood flow changed minimally (<10%) in all groups (data not shown). Hepatic sinusoidal insulin concentrations increased by a physiologic amount during the experimental period (150–300 min; Table 2Go), with no significant differences among the groups. Hepatic sinusoidal glucagon concentrations increased {approx}3-fold during the experimental period (150–300 min) compared with the basal period (90–120 min) and were similar in all groups (Table 2Go).


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  		TABLE 2. Hepatic plasma flows and hepatic sinusoidal insulin and glucagon concentrations1

 
The arterial plasma glucose concentration during the basal period and during the AA infusion period did not differ significantly among groups (Figure 2Go). The hepatic glucose load increased significantly during glucose infusion and reached 178 ± 10, 175 ± 6, and 172 ± 6 µmolkg–1min–1 in the EuAA, PeAA, and PoAA conditions, respectively. In the basal period, the animals of the 3 groups exhibited similar net hepatic glucose output at a rate of 10.2 ± 0.8, 8.8 ± 1.4, and 8.2 ± 0.5 µmolkg–1min–1 in the EuAA, PeAA, and PoAA conditions, respectively (Figure 2Go). During the AA infusion period, they shifted to net hepatic glucose uptake (NHGU). With a mean rate of –3.6 ± 1.5 µmolkg–1min–1 (between 210 and 300 min), NHGU in the PoAA group was significantly lower than in the EuAA (–7.7 ± 0.7 µmolkg–1min–1) and PeAA (–5.7 ± 0.9 µmolkg–1min–1) groups (Figure 2Go).


Figure 2
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  		FIGURE 2.. Portal amino acid infusion reduced net hepatic glucose uptake (NHGU). Amino acids were infused as described in Figure 1 and Table 1Go. Arterial, portal, and hepatic blood samples were drawn at the time indicated in the figure. Glucose concentrations were determined and NHGB was calculated as described in the Methods. Results represent the mean ± SE of 9 animals for each group. EuAA, eu-aminoacidemia with peripheral delivery; PeAA, hyper-aminoacidemia with peripheral delivery; PoAA, hyper-aminoacidemia with portal delivery. The statistical evaluation of the data was performed by a 2-way repeated-measures analysis of variance to test the group and time effects and time x group interaction. When significant, the Bonferroni test was used for post hoc analysis. Differences were considered significant when P < 0.05. Time effect, P < 0.0001; group effect, P < 0.0001; time x group interaction, P < 0.011. *PoAA significantly different from both EuAA and PeAA. PeAA not significantly different from EuAA.

 
Amino acid concentrations and arterioportal venous gradient
Hepatic AA loads were similar in the 3 groups (except for arginine) during the basal period (Table 3Go). Hepatic AA loads in the EuAA group were maintained at the basal values during the experimental period for each amino acid. Hepatic amino acid loads increased in the PeAA and PoAA groups when compared with the EuAA group (except for Ala, Arg, Cys, and Gly) but were similar in the PeAA and PoAA groups (except for Trp and Tyr; Table 3Go). During the AA infusion, the total hepatic load of AAs (sum of all AA loads) increased significantly but similarly in the PeAA and PoAA groups compared with the EuAA group (82.3 ± 4.3 and 82.5 ± 7.7 compared with 52.6 ± 3.4 µmolkg–1min–1, respectively). To maintain the matching of hepatic AA loads in the PeAA and PoAA groups, the portal infusion of AAs resulted in arterial AA concentrations lower than the ones generated with the peripheral AA infusion used in the PeAA group (except for Trp and Asp; Table 3Go). The PeAA and PoAA groups differed by the generation of a negative arterioportal venous gradient of AAs (ie, more negative when AAs were infused intraportally; Table 3Go). The arterioportal gradient in the PeAA group for most of the AAs was no different from that observed in the postabsorptive state (ie, the EuAA group).


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  		TABLE 3. Arterial and portal amino acid concentrations, hepatic amino acid load, and amino acid arterioportal gradient in the 3 experimental groups1

 
In the liver, the intracellular leucine concentration increased significantly in the PeAA and PoAA groups compared with the EuAA group (Figure 3Go). This increase ranged from 2 to 2.5 times depending on the liver lobe considered, but the same intracellular leucine concentration was observed whatever the route of delivery of the AAs (PeAA or PoAA; Figure 3Go).


Figure 3
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  		FIGURE 3.. Intracellular leucine concentration in the liver lobes of the dogs within the 3 experimental groups. EuAA, eu-aminoacidemia with peripheral delivery; PeAA, hyper-aminoacidemia with peripheral delivery; PoAA, hyper-aminoacidemia with portal delivery. At the end of the infusion period, liver samples were quickly removed and frozen in liquid nitrogen and stored at –80 °C until analyzed. Liver samples were prepared as described in the Methods, and leucine was determined as described in Donahue et al (19). The results represent the mean ± SE of 9 animals for each group. The statistical evaluation of the data was performed by a 2-way repeated-measures analysis of variance to test the group and lobe effects and lobe x group interaction. When significant, the Bonferroni test was used for post hoc analysis. Differences were considered significant when P < 0.05. Group effect, P < 0.0001; lobe effect, P < 0.036; no significant interaction. EuAA was significantly different from PoAA and PeAA; PeAA was not significantly different from PoAA.

 
Hepatic leucine balance and hepatic protein synthesis
During the experimental period, no significant differences in net hepatic leucine, valine, or isoleucine balance were seen with peripheral AA infusion. By contrast, portal AA infusion to create the same hepatic leucine, valine, and isoleucine loads increased net hepatic leucine, valine, and isoleucine uptake in comparison with PeAA (Figure 4GoA). Similar results were observed with other AAs, including tryptophan, lysine, glutamine, glutamate, and tyrosine (data not shown).


Figure 4
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  		FIGURE 4.. Arterial specific radioactivity of 14C leucine, net hepatic branched-chain amino acid balance, and net 14C leucine utilization. Amino acids were infused as described in the legend to Figure 1 and Table 1Go. EuAA, eu-aminoacidemia with peripheral delivery; PeAA, hyper-aminoacidemia with peripheral delivery; PoAA, hyper-aminoacidemia with portal delivery. Arterial, portal, and hepatic blood samples were drawn at the time indicated in the figure. Leucine and leucine specific activity were determined as described in the Methods. The results represent the mean ± SE of 9 animals for each group. The statistical evaluation of the data was performed by a one-way analysis of variance to test the group effect. The Bonferroni test was used for post hoc analysis. Values with different letters are significantly different, P < 0.05.

 
Arterial plasma leucine specific radioactivity was constant and similar among the 3 groups throughout the experimental period (210 to 300 min), and this confirms that a steady state of leucine kinetics was achieved in all groups (Figure 4GoB). Leucine-specific radioactivities were similar in portal plasma: 74.4 ± 3.9, 71.6 ± 4.2, and 71.3 ± 4.3 dpm/nmol in the EuAA, PeAA, and PoAA groups, respectively (mean values between 210 and 300min). Hepatic tracer balance showed results similar to the net hepatic leucine balance. Only the portal infusion of AAs was associated with increased leucine uptake into the liver compared with the EuAA condition (Figure 4GoC).

Liver protein synthesis was measured in the 7 lobes at 300 min (ie, after 180 min of AA infusion). Hepatic leucine specific radioactivities were 29.4 ± 1.9, 41.6 ± 2.8, and 36.8 ± 3.9 dpm/nmol in the EuAA, PeAA, and PoAA groups, respectively. Peripheral delivery of AAs did not increase hepatic protein synthesis significantly, whereas portal delivery stimulated the fractional synthesis in all liver lobes (Figure 5Go). The stimulatory effect of portal delivery of AAs was also found on exported hepatic proteins. Indeed, albumin synthesis was significantly increased by 30%, whereas peripheral AA infusion had no significant effect compared with EuAA (Figure 5Go).


Figure 5
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  		FIGURE 5.. Hepatic and albumin fractional protein synthesis rates. Amino acids were infused as described in the legend to Figure 1 and Table 1Go. EuAA, eu-aminoacidemia with peripheral delivery; PeAA, hyper-aminoacidemia with peripheral delivery; PoAA, hyper-aminoacidemia with portal delivery. Arterial, portal, and hepatic blood samples were drawn at times indicated in the figure. At the end of the infusion period, liver samples were quickly removed and frozen in liquid nitrogen and stored at –80 °C until analyzed. Liver samples were prepared as described in the Methods, and leucine and leucine specific activity were determined as described in Donahue et al (19). Liver protein synthesis was analyzed on the 7 hepatic lobes in each dog. Albumin synthesis was determined kinetically from 210 to 300 min. The results represent the mean ± SE of 9 animals for each group. For liver synthesis, the statistical evaluation of the data was performed by a 2-way repeated-measures analysis of variance to test the group and lobe effects and lobe x group interaction. When significant, the Bonferroni test was used for post hoc analysis. Differences were considered significant when P < 0.05. Group effect, P < 0.0001; lobe effect, P < 0.025; no significant interaction. PoAA was significantly different from EuAA and PeAA; PeAA was not significantly different from EuAA. For albumin synthesis, the statistical evaluation of the data was performed by a 2-way repeated-measures analysis of variance to test the group and time effects and time x group interaction. When significant, the Bonferroni test was used for post hoc analysis. Differences were considered significant when P < 0.05. No significant time effect; group effect, P < 0.0001; no significant interaction. PoAA was significantly different from EuAA and PeAA; PeAA was not significantly different from EuAA.

 
Signaling pathways
Because of its central role in mediating increases in protein synthesis, mTORC1 signaling was assessed in the livers of each group. As an index of alterations in mTORC1 signaling, the phosphorylation status of the downstream target, rpS6, on Ser235/236 and Ser240/244 was examined by Western blot analysis. This target was chosen because it was expected to be less subject to transient changes over such a long experimental protocol. As shown in Figure 6Go, AAs infused peripherally had no significant effect on phosphorylation of rpS6 on residues Ser240/244, whereas there was a trend (P = 0.07) for increased rpS6 phosphorylation on residues Ser235/236. In contrast, AAs infused portally led to a significant increase in phosphorylation of rpS6 at both Ser235/236 and Ser240/244 compared with EuAA values. In addition, portal AA delivery produced a significant (P < 0.05) increase in phosphorylation on Ser235/236 and tended (P = 0.1) to increase phosphorylation on Ser240/244 compared with peripheral delivery. Activation of mTORC1 was also monitored by assessment of the phosphorylation status of S6K1 as well 2 other proximal targets of the kinase, ie, 4E-BP1 and eIF4G. Although all 3 mTORC1 substrates showed the same pattern of increased phosphorylation as rpS6 in response to portal versus peripheral delivery of AAs, the magnitude of the change at the 180-min time point was less than that of rpS6 and was not significant. For example, phosphorylation of 4E-BP1, expressed as a percentage of the protein present in the hyperphosphorylated {gamma}-form relative to the total protein, was 59.5 ± 0.8%, 61.37 ± 0.7%, and 84.2 ± 3.8% for the control, AA infused peripherally, and AA infused portally conditions. Likewise, phosphorylation of eIF4G on Ser 1108, expressed in arbitrary units, was 0.27 ± 0.03, 0.36 ± 0.04, and 0.42 ± 0.1 for the control, AA infused peripherally, and AA infused portally conditions, respectively.


Figure 6
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  		FIGURE 6.. Amino acids infused portally, but not peripherally, activate mTORC1 signaling as indicated by increased phosphorylation of rpS6. Amino acids were infused as described in Figure 1Go and Table 1Go. EuAA, eu-aminoacidemia with peripheral delivery; PeAA, hyper-aminoacidemia with peripheral delivery; PoAA, hyper-aminoacidemia with portal delivery. At the end of the infusion period, liver samples were quickly removed and frozen in liquid nitrogen and stored at –80 °C until analyzed. Phosphorylation of rpS6 on (A) Ser235/236 and (B) Ser240/244 was measured by Western blot analysis as described in the Methods. In addition, samples were analyzed for eEF2 content to demonstrate equal loading of protein on the gel (see insert to panel A). eEF2 was used as a loading control because its expression did not change under the experimental conditions used in this study. The results represent the mean ± SE of 9 animals for each group. Representative Western blots are shown in the insets to each panel. The statistical evaluation of the data was performed by a one-way analysis of variance to test the group effect. The Bonferroni test was used for post hoc analysis. *P < 0.002 versus EuAA; {dagger}P < 0.05 versus peripheral amino acid condition.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Peripheral delivery of AAs to produce plasma concentrations seen postprandially did not stimulate hepatic protein synthesis significantly compared with euaminoacidemia, but synthesis of resident liver protein and albumin increased significantly (30%) with portal AA delivery. The stimulation of hepatic protein synthesis with portal delivery of AAs could not be attributed to differences in the hepatic AA load or in insulin, glucagon, or glucose concentrations. Thus, the route of delivery of AAs is critical for the stimulation of liver protein synthesis in adult dogs.

In support of our observation, human studies have shown that albumin synthesis is stimulated with oral meal feeding, whereas it is not responsive to intravenous nutrients (4, 12). Moreover, 20-96% of enterally administered AAs are utilized by the splanchnic bed, showing the crucial role of the splanchnic tissues in the distribution and availability of dietary AAs to peripheral tissues (21-24). Interestingly, recent studies conducted in pigs show that the dietary requirement for AAs (lysine, methionine, leucine, valine, isoleucine, and threonine) is lower with total parenteral nutrition than with enteral nutrition (21, 25-28), which is consistent with an increased utilization of dietary AAs by the gut and liver. Complementary results have shown that dietary AAs are the preferential source of substrate for hepatic protein synthesis (29-31). In the postprandial state, AA concentrations are higher in portal vein than in hepatic artery blood (32). However, a difference in AA availability could not explain our results, because the hepatic loads of AAs were carefully matched. It is possible that the portal delivery of AAs initiates a signal to the liver that stimulates hepatic AA utilization, ie, protein synthesis. Several studies conducted in our laboratory suggest the existence of an AA-initiated portal signal.

Previously, we carried out a series of studies to examine the role of AAs and their route of delivery (peripheral vein versus portal vein infusions) on NHGU in conscious dogs. With a fixed hormonal milieu (4x basal insulin; 1x basal glucagon) and a portal glucose infusion, portal gluconeogenic AA infusion reduced NHGU and net hepatic glycogen synthesis by 50% and 30%, respectively (33). This decrease could have originated from the hepatic AA load itself (ie, competition between substrates for hepatic uptake) or it could have been specific to the intraportal route of AA delivery. To answer this question (34), we studied a group of dogs under conditions identical to those in our previous studies except that the gluconeogenic AAs were delivered peripherally at a rate that would maintain the hepatic AA load equivalent to that in the previous studies. Peripheral delivery of AAs did not reduce NHGU initiated by portal glucose delivery. This observation is thus consistent with the hypothesis that intraportal delivery of AAs generates a signal that competes with or modulates the signal that enhances NHGU during portal glucose delivery. Similar conclusions can be drawn from the present experiments, because the portal delivery of all AAs also lowered NHGU when compared with peripheral AA infusion. In the study by Moore et al (33), with the peripheral AA infusion, the net hepatic uptake of glucose and AAs (in carbon equivalents) together equaled the net hepatic glycogen deposition and lactate release. With the portal AA infusion, only 70% of their uptake could be accounted for glycogen synthesis and lactate release. Our present study strongly suggests that at least a portion of the remaining 30% of carbon extracted by the liver was then redirected to protein synthesis. Taken together, our data indicate that a portal signal may activate liver protein synthesis. The portal concentrations of almost all of the AAs were statistically indistinguishable in the PoAA and PeAA conditions, which suggests that the negative arterioportal gradient of AAs initiates the signal.

Whether portal AAs modulate the activity of the autonomic nervous system to increase hepatic protein synthesis is unknown. However, the involvement of neuronal input in controlling liver glucose metabolism has already been shown (see reference 35 for a review), and Niijima et al (36, 37) have described hepatoportal sensors for 15 different AAs serving as stimulators or inhibitors of the vagal afferent discharge rate. Moreover, Watanabe et al (38) postulated that plasma protein synthesis is enhanced by vagal nerve stimulation.

The results of the present study show that portal, but not peripheral, delivery of AAs leads to a significant increase in phosphorylation of rpS6 on Ser235/236 and Ser240/244. Both sets of sites are phosphorylated by the rpS6 kinase S6K1 (39). Because mTOR phosphorylates S6K1 (40), an event that is critical for maximal activation of the kinase, this finding suggests that portal AA delivery increases mTORC1 signaling in liver to a greater extent than does peripheral delivery. Phosphorylation of S6K1 and 4E-BP1 was maximally increased 60 min after refeeding fasted rats and returned toward control values within 180 min (41). In contrast, phosphorylation of rpS6 was maintained through the 180-min time point. Therefore, because the liver samples in the current study were collected after 180 min of amino acid infusion, rpS6 phosphorylation should be a better indicator of mTORC1 activation.

Previous studies by our laboratory and others have provided evidence in support of the concept that albumin mRNA is translationally regulated in response to the availability of nutrients. Yap et al (42) showed that, after a 24-h fast in rats, albumin mRNA shifts out of polysomes and accumulates in 30-50S ribonucleoprotein complexes. Moreover, within 1 h of feeding a complete meal, the albumin mRNA redistributes back into polysomes. Studies in perfused rat liver (43) suggested that the translation of albumin mRNA is repressed by deficiency of essential AAs. Kuwahata et al (44), using acute liver injury in rats as an experimental model, showed that polysome-associated albumin mRNA increases in proportion to the content of branched-chain AAs during total parenteral nutrition. Finally, a cDNA microarray analysis of polysome-associated mRNAs over a time course after meal feeding to fasted rats showed translational control of albumin mRNA (43). Taken together, these data indicate that the mTORC1 signaling pathway is intimately involved in regulating albumin mRNA translation, which helps to explain the increase in albumin production observed in the PoAA condition.

Leucine modulates the activity of the signaling pathway leading to the initiation of protein translation (45-47). However, intracellular leucine concentrations were identical in the PoAA and PeAA livers, which suggests that the stimulation of the mTOR pathways in the liver is also under the control of signal inputs that are different from the leucine pathways and initiated specifically by the portal AA infusion. In addition to actions on the mTOR signaling pathway, portal versus peripheral infusion of AAs might affect liver protein synthesis via other mechanisms, such as the metabolic sensors AMP-activated protein kinase or general control nonderepressible 2 kinase (GCN2; 48).

Although mTORC1 is a central mediator of the increase in protein synthesis engendered by increased provision of amino acids, other signaling pathways may also be involved in the response. For example, GCN2 is activated in response to deprivation of essential amino acids (49). On activation, GCN2 phosphorylates the {alpha}-subunit of eIF2 leading to inhibition of the first step in translation initiation. Thus, eIF2{alpha} phosphorylation is increased in livers from rats fed a diet lacking either Trp or Leu compared with animals fed a complete meal or a diet lacking the nonessential amino acid Gly (50). However, in the present study, there was no significant difference in eIF2{alpha} phosphorylation in livers from dogs infused with amino acids peripherally compared with portally (0.82 ± 0.04 versus 0.79 ± 0.08 arbitrary units, respectively). The basis for the lack of change in eIF2{alpha} phosphorylation in the present study is unknown, but is likely related to amino acids being provided in a balanced manner (ie, one or more essential amino acids were not limiting). In this regard, in previous studies (51), no change in eIF2{alpha} phosphorylation was observed in liver in response to an overnight fast or after refeeding overnight-fasted animals.

In conclusion, portal delivery of AAs stimulated hepatic synthesis of resident and exported proteins, whereas peripheral delivery of AAs did not, despite similar AA concentrations within the liver. Our results suggest that a portal signal was generated with the portal infusion of AAs that was necessary to fully stimulate hepatic AA utilization and hepatic protein synthesis. Thus, enteral nutrition may be preferred over parenteral nutrition in stimulating or maintaining liver protein synthesis in humans. In contrast, an enhanced splanchnic extraction of AAs may be deleterious in sustaining AA provision to peripheral tissues such as skeletal muscle. This has been suspected, for example, to be responsible for the defect in the postprandial stimulation of muscle protein synthesis during aging. When muscle protein synthesis is of key importance, our findings suggest that a peripheral infusion of AAs may be advantageous over a portal AA infusion to spare AAs.


   ACKNOWLEDGMENTS
 
The authors' responsibilities were as follows—DD: planned and implemented the study, analyzed statistics, interpreted data, and wrote the manuscript; SRK: sample analysis and critical revision of the manuscript; LSJ: sample analysis and critical revision of the manuscript; ADC: critical revision of the manuscript; DR: analyzed statistics and critical revision of the manuscript; CAD: sample analysis; MCM: study design and critical revision of the manuscript. None of the authors had a conflict of interest.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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Received for publication March 19, 2008. Accepted for publication June 17, 2008.





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